The present invention relates to 3-indocyanine green-acyl-1,3-thiazolidine-2-thiones useful for fluorescent labeling agents and the like.
Polymethyne dyes absorb light in the near infrared region (600 to 1200 nm) and emit fluorescence (Patonay, G. et al., Anal. Chem., 1991, 63, 321A). They are characterized by high molecular absorbance and very high fluorescent emission. One of these dyes, indocyanine green (ICG) (U.S. Pat. No. 2,895,955; 1959), has an excellent feature of extremely low interference by absorption scattering in the near infrared region and fluorescence of a molecule in a living body, and has been clinically used for diagnosis of liver functions. Accordingly, derivatives of the aforementioned fluorescent compound are expected to be useful as fluorescent labeling agents.
It has been known that proteins such as bovine serum albumin (BSA) and immunoglobulin G (IgG) can be labeled with fluorescence by using the aforementioned fluorescent compound per se or its derivative (Southwick P. L. et al., Cytometry, 1990, 11, 418; Ernst, L. A. et al., Cytometry, 1989, 10, 3; Mujumdar, R. B. et al., Cytometry, 1989, 10, 11; Mujumdar, R. B. et al., Bioconjugate Chem., 1933, 4, 105; Ito, S. et al., Bioorg. Med. Chem. Let., 1995, 5, 2689). In these reports, it is explained that a protein labeled with fluorescence can be used for ultramicroanalysis by fluorescence measurement (Mujumdar, R. B. et al., Bioconjugate Chem., 1933, 4, 105), and that the protein can be a diagnostic reagent for very small stomach cancers (Ito, S. et al., Bioorg. Med. Chem. Let., 1995, 5, 2689).
Nagao et al. elucidated some features in aminolysis of various 3-acyl-1,3-thiazolidine-2-thione (ATT) derivatives by utilizing active amide structures, and reported that the ATT moiety selectively reacts with an amino group (Nagao, Y. et al., J. Chem. Soc., Perkin Trans., 1, 1984, 2439; Nagao, Y. et al., Heterocycles, 1982, 17, 537).
An object of the present invention is to provide novel compounds useful as fluorescent labeling agents. In particular, the object of the present invention is to provide compounds which have high fluorescence intensity in the near infrared region and can function to group-selectively and efficiently label proteins and oligopeptide compounds. The inventors of the present invention made intensive studies to achieve the aforementioned objects. As a result, they found that indocyanine green amide derivatives of 1,3-thiazolidine-2-thione have the aforementioned features and are extremely useful as fluorescent labeling agents. The present invention was achieved on the basis of the above findings.
The present invention thus provides compounds represented by the following formula (I): 
wherein R represents a lower alkyl group which may be substituted; n represents an integer of from 1 to 10; and X represents an anion species. According to preferred embodiments of this invention, there are provided the aforementioned compounds wherein R is a C1-6 alkyl group which may have one sulfonic acid group, and n is an integer of from 4 to 6; the aforementioned compounds wherein R is a C1-6 alkyl group, and n is 5; and the aforementioned compound wherein R is ethyl group, and n is 5. As a preferred embodiment of this invention, there is provided 2-[7-(1,3-dihydro-1,1-dimethyl-3-ethylbenz[e]indolin-2-ylidene)-1,3,5-heptatrienyl]-1,1-dimethyl-3-[[6-[(1,3-thiazolidine-2-thion)-1-yl]-6-oxo]hexyl]-1H-benz[e]indolium chloride.
According to another aspect of the present invention, there are provided a fluorescent labeling agent which comprises the compound represented by the aforementioned formula (I); a medicament for diagnosis which comprises the compound represented by the aforementioned formula (I), and is preferably used for fluorescence imaging method; and a medicament which comprises the compound represented by the aforementioned formula (I) and is preferably used for cancer treatment by using a laser, more preferably for endoscopic cancer treatment by using a near infrared laser.
According to a further aspect of the present invention, there are provided a use of the compound represented by the aforementioned formula (I) for the manufacture of the aforementioned medicament; a method of immunochemical staining of a living tissue by using the compound represented by the aforementioned formula (I); a method for diagnosis by fluorescence imaging method with the compound represented by the aforementioned formula (I), preferably a method for diagnosis of a cancer; and a method for endoscopic treatment of a cancer with a laser, preferably a near infrared laser, by using the compound represented by the aforementioned formula (I).
In the aforementioned formula (I), R represents a lower alkyl group which may be substituted; n represents an integer of from 1 to 10; and X represents an anion species. As the lower alkyl group, a straight chain or branched chain alkyl group having carbon atoms of from 1 to about 6 can be used. More specifically, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, sec-butyl group, tert-butyl group or the like can be used. The symbol xe2x80x9cnxe2x80x9d is preferably an integer of from 4 to 6, more preferably 5. The kind of X is not particularly limited. For example, as an monovalent anion species, a halogen ion such as chlorine ion, bromine ion, or iodine ion can be used.
When the lower alkyl group represented by R has substituents, the number, the kind, and the position of the substituents are not particularly limited, and examples of the substituents include sulfonic acid group, hydroxyl group, carboxyl group, halogen atoms (may be any of fluorine atom, chlorine atom, bromine atom, or iodine atom), lower alkoxyl groups (e.g., methoxy group, ethoxy group and the like), amino group, mono- or di(lower alkyl)amino groups, keto group, lower alkanoyl groups and the like. The lower alkyl moiety in the aforementioned substituents may have one or more kinds of these substituents. R is preferably an unsubstituted lower alkyl group, or an alkyl group substituted with one sulfonic acid group, and more preferably, ethyl group can be used.
The compounds of the present invention may sometimes have one or more asymmetric carbon atoms depending on the kind of substituents, and isomers such as optical isomers and diastereoisomers based on one or more asymmetric carbon atoms may sometimes exist. Any of such isomers, any mixtures of the isomers, racemates and the like falls within the scope of the present invention. In addition, when the compound of the present invention has an alkyl group having an acidic substituent as group xe2x80x9cRxe2x80x9d, for example, sulfonic acid group and the like, the compound may sometimes form an intermolecular twitter ion without need of an anion species represented by Xxe2x88x92. It should be understood that such compounds also fall within the scope of the present invention. A compound which forms a twitter ion in the molecule may sometimes have reduced ionicity and fails to have sufficient water solubility. Accordingly, when an alkyl group having a sulfonic acid group or the like is used as R, the sulfonic acid group is desirably in the form of a metal salt such as sodium sulfonate. Examples of the combination of a metal ion which forms the metal salt and Xxe2x88x92 preferably include, for example, NaI, KI, NaCl and the like.
A preferred compound among those of the present invention includes, for example, 2-[7-(1,3-dihydro-1,1-dimethyl-3-ethylbenz[e]indolin-2-ylidene)-1,3,5-heptatrienyl]-1,1-dimethyl-3-[[6-[(1,3-thiazolidine-2-thione)-1-yl]-6-oxo]hexyl]-1H-benz[e]indolium chloride (herein sometimes referred to as xe2x80x9cICG-ATTxe2x80x9d). However, the compound of the present invention is not limited to the aforementioned specific compound.
Methods for preparation of the compounds of the present invention are not particularly limited, and those prepared by any methods of preparation fall within the scope of the present invention. As an example of the preparation of the compound of the present invention, the preparation of ICG-ATT is shown in the scheme set out below. In addition, details of the preparation will be shown in Examples of the present specification. Accordingly, a person skilled in the art can easily prepare the compounds of the present invention which fall within the aforementioned formula (I) by referring to the following scheme and specific explanations in Examples, and appropriately choosing starting materials, reagents and reaction conditions, and if necessary, by adding appropriate alteration or modification to these methods (in the scheme, Et represents ethyl group; Me, methyl group; WSCD, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; and, DMAP, dimethylaminopyridine). 
The compound of the present invention has properties that the compound is substantially non-fluorescent under the normal solar light or a fluorescent lamp, whilst the compound emits extremely strong fluorescence when being irradiated by the near infrared light. In addition, the compound of the present invention has excellent solubility in water, and is featured to react selectively and efficiently with amino group existing in amino acids, oligopeptides, proteins or the like to give acylated compounds. Accordingly, the compound of the present invention is useful as a fluorescent labeling agent for amino acids, oligopeptides, proteins, fats, saccharides and the like.
For example, the existence of cancer cells or cancer tissues can be verified by labeling an anti-tumor antibody with the compound of the present invention, and then allowing the labeled antibody to contact with a tissue or an organ. For diagnosis, a tissue section or the like may be fixed by an appropriate method such as the paraffin method and observed under a microscope. A living tissue can be immunochemically stained and observed with an endoscope. Recently, various fluorescence imaging methods using a near infrared fluorescent substance have been proposed (e.g., Japanese Patent Unexamined Publication (KOKAI) Hei. No. 9-309845; J. Neurosurg., 87, pp. 738-745, 1997; Medical Electron and Bioengineering (Iyodenshi to Seitaikogaku), 34, pp. 316-322, 1996 and the like), and the compound of the present invention can be utilized as a medicament for diagnosis utilizing the fluorescence imaging technique.
Moreover, a method of endoscopic treatment of an early gastric cancer by using a high power semiconductor laser with indocyanine green has been proposed (Gastroenterological Endoscopy, 39, 1753-1765, 1997; Gastroenterological Endoscopy, 39, 1766-1774, 1997). This method can elevate laser absorption in a tissue with the aid of indocyanine green and promote the heat clotting or transpiration of tissues, especially transpiration, and is expected to be useful for treatments of early cancers. The compound of the present invention can also be used for the aforementioned laser treatment. As the laser, high power semiconductor lasers in the near infrared region are suitable, and examples of a cancer to be treated include, for example, stomach cancer, esophageal carcinoma, duodenal carcinoma, rectum cancer and the like. Cancers curable by percutaneous endoscopic operations may also be treated.
When the compound of the present invention is used as a medicament for the aforementioned diagnosis or therapeutic treatment, the compound is preferably prepared in the form of a pharmaceutical composition by using one or more kinds of additives for pharmaceuticals. Pharmaceutical compositions in the form of a solid or a liquid or the like can be prepared, for example, by using appropriate additives for pharmaceuticals such as buffering agents, solubilizing aids, pH adjusters, excipients, antiseptics and the like. The form of the pharmaceutical composition suitable for diagnosis or therapeutic treatment and the preparation thereof can be appropriately chosen by a person skilled in the art. Uses of the compound of the present invention are hereinbefore specifically explained. However, the uses of the compound of the present invention are not limited to the aforementioned examples.